Unlocking the Mysteries of Muscle Contraction: A Breakthrough in Ryanodine Receptor Structure

Ever wonder how our muscles work when we move? It all comes down to calcium—a key player, released from storage spaces within our muscle cells called the sarcoplasmic reticulum (SR), that regulates the muscle contraction. The crucial protein allowing calcium release is the type 1 ryanodine receptor (RyR1), an ion channel of enormous proportions (the largest known so far) that is embedded in the SR membrane.
RyR is more than just a protein; it’s a critical piece of the puzzle in understanding diseases like muscular dystrophy and heart failure. Plus, RyR serves as a potential pharmacological target, opening avenues for therapeutic interventions in muscle-related disorders.
Yet, working with such bulky proteins in its native state – within the cell membrane- is a challenging subject. Typically, the steps needed to resolve the structure of proteins normally involve using detergents and harsh conditions that disrupt the native state of a protein.
However, why is it crucial to comprehend the structure of proteins in their native state?? Because it enables us to mimic the conditions in living cells and gain valuable insights into their physiological relevance, their interaction with other molecules, exploring disease mechanisms, and facilitating drug development, among others.
So far, it has been used cryogenic electron microscopy (cryo-EM), an imaging technique that freezing samples and using electrons instead of light to reveal the protein’s intricate 3D structure (i.e., protein’s shape, how its various parts are arranged, and the interactions between its amino acids). While the 3D arrangement of the RyR1 protein had been solved at a very detailed level (referred to as the “High-resolution structure”), the configuration in its native state was still unknown.
In the pursuit to unravel the structure of proteins within native or near-native membranes, a recent breakthrough has provided a high-resolution view of RyR1.
Departing from traditional detergent-based methods used to solubilize and stabilize membrane proteins, Dr Melville Zephan, CUIMC postdoc, and colleagues adopted a gel-filtration approach with on-column detergent removal, they’ve captured RyR1 within liposomes—tiny bubbles mimicking cell membranes (Figure 1).

Figure1. Flow diagram illustrating protein-incorporated liposomes.
RyR1 from rabbit skeletal muscle was homogenized and purified using ion-exchange chromatography, a technique that separates compounds based on their electric charge. Liposomes, which are tiny vesicles made of self-assembly of lipid bilayers in water, were formed using gel filtration. This process helped incorporate RyR1 into liposomes and remove any excess or empty liposomes. (Adapted from Melville Z. Cell Press 2021)

This novel technique maintains a more natural environment, allows to purify membrane proteins and remove detergent molecules in excess, offering a clearer glimpse into how RyR1 functions (Figure 2).

Figure2. RyR1 representation in native and near-native membranes incorporated in liposome after gel filtration. (Adapted from Melville Z. Cell Press 2021)

Their study reveals how RyR1 behaves in its natural environment, forming a network of channels within cell membranes. While their investigation in liposomes gives only a partial view of this complexity (without seeing how molecules interact), it sets the stage for future investigations. This approach, extending beyond RyR, offers a blueprint for studying membrane proteins in native conditions.

In summary, this work successfully captured the intricate structure of RyR1 in liposomes opening doors to a better understanding of muscle physiology and offers a sharper lens for exploring the complex world of membrane proteins.

For more details on this research, refer to the article: “High-resolution structure of the membrane-embedded skeletal muscle ryanodine receptor”.

https://www.sciencedirect.com/science/article/pii/S0969212621002963?via%3Dihub

Reviewed by: Trang Nguyen, Carlos Diaz, Erin Cullen

 

Unveiling the secrets of pain: decoding the structure of a human receptor for effective relief

Pain is an essential sensation for the survival of organisms. It acts as a protective mechanism, signaling potential harm and prompting animals to recognize noxious stimuli for avoiding future harm. For example, individuals unable to feel pain sensations (a rare congenital disease) seem advantaged at first glance; however they are actually at a high risk of unknowingly injuring themselves. While acute pain serves a crucial role in preserving well-being, chronic or persistent pain can severely impact the quality of life. Unfortunately, the mechanisms underlying the transition from acute to chronic pain remains poorly understood, making diagnosis and treatment challenging. Although opioids are commonly used to manage chronic pain, they carry the risk of addiction and interference with normal brain activity. As an alternative, targeting directly the receptors involved in pain pathways, such as the transient receptor potential (TRP) family, offers a promising avenue for developing analgesic therapies.

A recent study (https://www.nature.com/articles/s41467-023-38162-9) led by Dr. Arthur Neuberger, a postdoctoral research scientist in the department of Biochemistry and Molecular Biophysics within Dr. Alexander Sobolevsky’s lab, investigated the structure and inhibition mechanism of human TRPV1 and its interaction with a potential analgesic compound known as SB-366791.

TRPV1, a receptor found on nerve cells, plays a pivotal role in sensing pain and heat. It is a temperature-sensitive TRP ion channel, commonly referred to as the vanilloid receptor 1 or capsaicin receptor (pungent compound from chili pepper). TRPV1 functions as an ion channel involved in pain sensation, becoming activated in response to noxious signals. When activated, TRPV1 opens, leading to a decrease in the electrical resistance of the cell membrane and the subsequent flow of ions. If the strength of the noxious signal and the response of the ion channel are sufficient, a change in the membrane potential occurs, allowing the signal to be transmitted from the peripheral nervous system to the spinal cord and ultimately to the brain (Figure 1).

Figure 1. Overview of the pain-processing pathway. When exposed to noxious stimuli, such as the burning sensation of a flame on the skin or the heat sensation of capsaicin on the tongue, TRPV1 ion channels are activated in the peripheral nervous system. The resulting heat signal is transmitted through nerve fibers and follows spinal pathways until it reaches the brain, where it triggers the generation of a pain response. Figure created using Biorender.

The study, published in Nature Communications, significantly advanced our understanding of the three-dimensional structure of TRPV1, providing crucial insights into its functional properties. The researchers employed advanced techniques, such as cryo-electron microscopy (cryo-EM), to determine the precise arrangement of human TRPV1. Additionally, whole-cell patch clamp electrophysiology (technique that measures the electrical signals as flow of ions across the cell membrane providing information on how the cell responds to certain stimuli or drugs) was used to propose the inhibition of TRPV1 by SB-366791. These cutting-edge techniques provided valuable insights into the architecture and functional characteristics of TRPV1 (Figure 2). Through their investigations, the researchers successfully identified the specific binding site of SB-366791 on the vanilloid site of TRPV1, utilizing cryo-EM and mutagenesis techniques. This discovery suggests that the analgesic compound may exert its effects by selectively inhibiting TRPV1 activity. Furthermore, the study revealed the structural changes that occur within TRPV1 upon binding to SB-366791. These structural alterations affect the conformation of the protein, demonstrating that even in the closed state of TRPV1, SB-366791 can bind and stabilize the closed conformation. This mechanism has the potential to reduce pain signaling. Understanding these structural modifications is important for the development of novel analgesic drugs targeting TRPV1.

Traditionally, pharmacological and physiological investigations of TRPV1 have primarily focused on rodent and squirrel orthologues. However, since the TRPV1 channel plays a central role in almost every aspect of human physiology and disease, including pain and temperature perception, the recent study by Neuberger and colleagues is significant. By specifically examining the human TRPV1 structure and its interaction with SB-366791, this research opens up new possibilities for the development of effective analgesic therapies and sets the stage for future investigations in the field of pain management.

Figure 2. Overview of cryo-electron microscopy (cryo-EM) and the structure of human TRPV1 in complex with the inhibitor SB-366791. In cryo-EM, a beam of electrons is directed at a frozen solution containing the protein of interest. The resulting electrons pass through a lens system, creating a magnified image on a detector. From this image, a three-dimensional model of the protein’s structure is generated. The structure of hTRPV1 in complex with SB-366791 is visualized from two perspectives: parallel to the membrane and extracellularly. In the images, the TRPV1 subunits are color-coded as green, yellow, pink, and blue, while the lipids are represented as purple sticks. (Figure modified from Neuberger et al., 2023)

Reviewed by: Maaike Schilperoort, Giulia Mezzadri and Trang Nguyen

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